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Alternative ligands for measurement and purification of ecdysteroid receptors in Drosophila Kc cells.

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Archives of Insect Biochemistry and Physiology 25-33 (1986)
Alternative Ligands for Measurement and
Purification of Ecdysteroid Receptors in
Drosophila K, Cells
Becky A. Sage, Denis H.S. Horn, Thomas M. Landon, and John D. O’Connor
Department of Biology, University of California, Los Angeles (B.A.S., ‘EM.L., J. D.O.); Division
of Applied Organic Chemistry, C.S.I.R.O., Melbourne, Victoriu, Australia (D.H.S.H.)
An ecdysteroid-binding protein has been demonstrated in nuclear and
cytosolic extracts of cultured Drosophila cells (K, cells). Attempts t o purify
the binding moiety have been hampered because of the small number of
binding sites (ca 1,00O/celI) and sensitivity of the ecdysteroid binding moiety
toward salt (dissociation at 150 mM). Recently 3~~[~H]kaladasterone
A have been synthesized. The binding kinetics of these
two ecdysteroids are similar t o ponasterone A. Photoaffinity labeling of the
ecdysteroid receptor in Kc cells was attempted using the 3~x[~H]kaIadasterone, and standard protein chromatographic techniques have been
used t o examine the 3~~[~H]muristerone
A-receptor complex, which is less
sensitive to salt dissociation. Attempts t o characterize the protein t o which
these ligands have been attached will be discussed.
Key words: photoaffinity labeling, kaladasterone, muristerone A
For the past several years, the & cell line of Drosophila melunogaster,
established by Echalier and Ohanessian [l], has been used as a model system
to study ecdysteroid action. There are four particular advantages inherent in
using an established insect cell line to study ecdysteroid action. The first is
the availability of large quantities of biological material. Two additional advantages are that the & cells do not metabolize ecdysteroids, nor do they
contain any endogenous hormone. This means that one can expose the &
cells to ecdysteroids of known concentration and have the dosage of hor-
Acknowledgments: We thank Dr. Maria A. Tanis for her efforts in synthesizing the precursor
for the radioligand, [3H]ponasteroneA. This work was supported by a grant from the National
Science Foundation.
Address reprint requests to Dr. John D. O’Connor, Department of Biology, University of
California, 405 Hilgard Avenue, Los Angeles, CA 90024.
0 1986 Alan R. Liss, Inc.
mone remain constant over a period of days. A final advantage of the K, cell
line is that the cells do show characteristic responses when exposed to
physiological concentrations of ecdysteroids. These responses have been
documented elsewhere, and include G2 arrest 121, changes in morphology
[3],increased intercellular adhesion [4],and induction of acetylcholine esterase [5], dopa decarboxylase [6], and ecdysteroid-inducible polypeptides 18.
Such responses are associated with the presence of an intracellular binding
moiety or ecdysteroid receptor.
In addition to its association with biological responses, an intracellular
binding protein must fulfill three kinetic criteria: Binding must be saturable,
specific, and of high affinity. The characterization of these three criteria for
the ecdysteroid receptor present in K, cells has been previously documented
[8].Using a radioligand of high specific activity ([3H]ponasteroneA, structure
C, Fig. 1, SA* 106 Cilmmol), an ecdysteroid binding moiety was obtained
from both cytosolic and nuclear preparations of K, cells not previously
exposed to hormone. This binding moiety demonstrated saturability, and
specificity for ponasterone A (Kd 3 nM), 20-OH-ecdysone (Kd 200 nM),
and ecdysone (Kd > 10 pM). These affinities correlated precisely with the
ecdysteroids' respective biological activities and, in the case of the latter two
ligands, with their physiological concentrations. Similar data have been obtained for imaginal discs [9].
Ho 0
Fig. 1. Ecdysteroid structures. I, ecdysone; 11, 20-OH-ecdysone, the molting hormone; Ill,
ponasterone A; IV, rnuristerone A.
*Abbreviations: HPLC = high-performance liquid chromatography; Kd = dissociation constant; SA = specific activity.
Purification of Ecdysteroid Receptors
When the hydrodynamic characteristics of this ecdysteroid receptor were
examined, it was found that the binding moiety present in both cytosolic and
nuclear preparations of K, cells sediments at 6 S. A 4 S sedimentation value
could also be obtained for the cytosolic receptor, but this occurred only if the
cells were frozen at -20°C prior to their homogenization and the preparation
of a high-speed cytosol.
The presence of the ecdysteroid binding moiety in both cytosolic and
nuclear preparations suggests that the ecdysteroid receptor is not specifically
localized within K, cells. Moreover, the data indicate that there exists within
K, cells a resident population of nuclear receptors whose number does not
significantly increase upon exposure to hormone [8]. The existence of a
significant population of resident nuclear receptors has also been documented in imaginal discs [lo], where approximately 95% of the receptor
isolated from third instar imaginal discs is localized in the nuclei of the
imaginal disc cells. This nuclear localization of the ecdysteroid receptor in
imaginal discs might represent its normal or regular residence or the translocation of receptor into disc nuclei by previous exposure to hormone. Support for the former hypothesis has been presented previously [8].
This observation of the nuclear localization of the ecdysteroid receptor in
hormonally naive cells leads one to propose the revised paradigm for ecdysteroid action shown in Figure 2. Briefly, after 20-OH-ecdysone enters the
cell, it enters the nucleus and binds to a receptor. This hormone-receptor
complex then interacts with the DNA to induce new RNA transcripts. The
hormone might also bind to receptors present in the cytosol, but the exact
role of these complexes has not as yet been identified.
Induced Prolein Synthesis
Fig. 2. Paradigm of ecdysteroid mode of action. The arthropod molting hormone, 20-OHecdysone (E), enters the target cell by diffusion and travels into the nucleus where it binds to
a receptor molecule (R). In the nucleus, the ecdysteroid receptor complex interacts with the
DNA to induce new RNA transcripts. Ecdysteroid receptor molecules may also be present in
the cytosol (stippled R) and able t o bind free ecdysteroid. Functions for these cytosolic
ecdysteroid receptor complexes have not as yet been clearly identified.
To understand fully the mode of action of the ecdysteroid-receptor complex, one must have the ecdysteroid receptor purified. Several difficulties
arise as one attempts to use standard purification techniques for this protein
binding moiety. The first difficulty is the fact that ecdysteroid receptor concentrations are very small. It is estimated that there are 1,000-1,500 receptorslcell. This receptor number is tenfold less than that found in most other
steroid systems [11,12]. The second difficulty is that the binding activity of
unloaded receptor is inherently labile when subjected to standard protein
purification techniques. Therefore, the ecdysteroid receptor needs to remain
loaded with hormone during most manipulations. The fact that the steroid
hormone complex has a relatively rapid dissociation constant at both 4°C
and 22°C [S] makes the necessity for constant loading of the receptor even
more difficult to maintain. A final difficulty, unique to the ecdysteroid system, is that the binding of the ecdysteroid to its receptor is salt-sensitive.
Using [3H]ponasteroneA as a radioligand, >50% of the bound ligand dissociates during a 30-min incubation with 150 mM salt at 24°C. Such rapid
dissociation in the presence of relatively low salt concentrations precludes
the use of standard ion exchange chromatography techniques and HPLC
protein separations that require high salt buffers.
Our laboratory has tried to circumvent these difficulties by attempting to
link covalently a radiolabeled ecdysteroid to its receptor by means of photoaffinity labeling. Although the conjugated 7-ene-6-one system, common to
the ecdysteroids (absorption maximum 242 nm), is photoreactive, we have
been unable to demonstrate significant covalent binding of either ponasterone A, 20-OH-ecdysone, or muristerone A (see Fig. 1for structures) to the
ecdysteroid receptor. Similar difficulties have also been documented for other
steroid receptor systems [13,14]. However, it has been found that steroid
ligands, which contain a conjugated diene-one functional group, are generally more photoreactive [14,15]. This enhanced photoreactivity can be attributed partially to the fact that the absorption maximum of the conjugated
diene-one system is 300 nm. This means that one should be able to irradiate
ecdysteroid receptor preparations more effectively using wavelengths of light
>320 nm. In doing so, one decreases the possibility that the protein population will undergo photodegradation during the photolysis procedure.
One biologically active ecdysteroid that contains this conjugated dieneone system is kaladasterone [16]. When unlabeled kaladasterone is incubated
with a nuclear receptor preparation obtained from K, cells [S], the data shown
in Figure 3 are obtained. The specific, rapid photoinactivation of receptor
binding activity during UV irradiation of the kaladasterone receptor complex
can be explained in one of two ways. Either the irradiation is causing the
kaladasterone to become bound covalently to the receptor or the irradiation
causes kaladasterone to destroy the ability of the receptor to bind the radioligand, [3H]ponasteroneA, following the irradiation procedure.
To determine which of these events is occurring, the kaladasterone must
be radiolabeled. A protocol for the synthesis of 3a[3H]kaladasterone from
Purificationof Ecdysteroid Receptors
uv Irradiation Time, minutes
Fig. 3. Photoinactivation of K, ecdysteroid binding moieties. Nuclear ecdysteroid receptor
preparations [8] were incubated with saturating amounts of unlabeled 20-OH-ecdysone (1
pM) or kaladasterone (100 nM). Receptor preparations were then irradiated for given periods
of time (Osram HBO-200W mercury lamp with glass filter, h > 320 nm, 4OC). Aliquots of the
irradiated receptor preparations were then incubated with high salt (KCI concentration
400 mM, 30 rnin, 24OC) to dissociate any noncovalent binding of ecdysteroid to the receptor.
The samples were then centrifuged through Sephadex C-25 to remove free ecdysteroid and
the KCI. The degree of photoinactivation of the ecdysteroid receptor was measured by
labeling the samples with [3H]ponasteroneA (60 min, 24OC, 1 nM) and determining bound
ligand using a dextran-coated charcoal assay [8]. Ecdysteroids present during photolysis: 0,
20-OH-ecdysone; A,kaladasterone; 0 ,control, no ecdysteroids present.
muristerone A was developed by one of us (D.H.S.H.) and is briefly outlined
in Figure 4. Following the final step of mild base hydrolysis, the
[3H]kaladasterone was purified on reverse-phase HPLC (Waters RCM-100,
Radial-Pak CI8 pBondapak lop; 55% methanol in water; 2.0 mllmin; elution
time of kaladasterone standard was 6 min 45 sec) and its specific activity
determined to be -4 Cilmmol. Saturation kinetics of a nuclear receptor
preparation incubated with [3H]kaladasteroneare shown in Figure 5. The Kd
from the Scatchard [17l analysis of these data was calculated to be 15.0 nh4.
In addition, the kaladasterone was shown to have biological activity in the
Kc cell line by inducing a G2 arrest in the cell cycle.
Irradiation of a [3H]kaladasterone nuclear receptor preparation initially
resulted in the photodegradation of the ecdysteroid receptor’s binding capability. The only condition that was effective in reversing this destruction of
receptor was to perform the photolysis under a nitrogen atmosphere. Under
a nitrogen atmosphere, radioactivity was found associated with TCA-precipitable protein (Table 1).However, if these proteins are separated using HPLC
anion exchange chromatography, the results shown in Figure 6 are obtained.
The absorbance and radioactivity profiles of the eluting proteins show that
the [3H]kaladasterone is being covalently cross-linked to a number of different protein species. Similar results of nonspecific photoaffinity labeling are
HO 0"
Muristerone A
3-Keto-Muristerone A
10% Aqueous CH,COOH
(-64% y i e l d )
3H-Kaladosterone. nM
Fig. 4. Outline of the syntheses of 3c~[~H]rnuristerone
A and 3c~[~H]kaladasterone
rnuristerone A.
Fig. 5. [3H]Kaladasteronesaturation of nuclear receptor preparation from K, cells. Aliquots
of a Kc nuclear extract [8] were incubated with increasing concentrations of [3H]kaladasterone
(75 rnin, 24OC). Bound ligand was determined by duplicate dextran-coated charcoal assays
[8]. Nuclear receptor preparations were incubated with either [3H]kaladasterone ( 0 )or
[3H]kaladasterone with 100-fold excess ponasterone A (A). Saturable binding of
[3H]kaladasteronewas obtained by subtraction of these two curves (0).Inset shows Scatchard [I71 analysis of saturation binding data.
Purification of Ecdysteroid Receptors
Time ( m i d
Fig. 6. HPLC separation of photoaffinity labeled proteins present in a K, nuclear receptor
preparation. A K, nuclear extract, in 50 mM potassium phosphate buffer, was incubated with
[3H]kaladasterone (50 nM), placed into a glass reaction vessel, and equilibrated with nitrogen
gas for 20 min. The sample was then photolyzed for 20 min under an N2 atmosphere (CanradHanovia medium pressure, mercury-vapor lamp, 450 W; h > 320 nm; 4OC). A portion of the
sample was then analyzed by DEAE anion exchange HPLC chromatography. (Chromatographic conditions: Pharmacia Mono Q HR 5/5 anion exchange column; 1 mllmin; elution
buffer at time 0 was 50 mM Tris, pH 8.0, followed at 3 rnin by a 10 min gradient of 0 to 350
m M KCI in 50 mM Tris, and at 18 min by 1.0 M KCI in 50 m M Tris.) Absorbance was monitored
Fractions of 0.5 ml were collected and monitored for radioactivity (0-0).
at 280 n m (-).
TABLE 1. Photoaffinity Labeling of a Nuclear Extract From Kc Cells
K, cell linea
(dpmi100 fig)
'&-WT designates a receptor-positive cell line; K,-BR - designates a receptor-negative cell
bRadioligand abbreviations: [3H]KAL is [3H]kaladasterone, [3H]PNA is [3H]ponasterone A.
'Experiments in which excess radioligand was removed prior to irradiation procedure.
also observed when free kaladasterone is removed from the nuclear receptor
preparation prior to the irradiation procedure. These data indicate that
[3H]kaladasterone is a possible photoaffinity ligand, but the high degree of
nonspecific covalent binding precludes using the ligand on a crude receptor
The synthesis of radiolabeled [3H]kaladasteronealso provided our laboraA. In early experitory with an additional radioligand, 3~t[~H]muristerone
ments with muristerone A, it was discovered that this particular ligand,
800 700 -
600 C
E 400-
200 100 -
Fig. 7. HPLC anion exchange chromatography of K, 4 S cytosol receptor preparation. Cytosol
receptor was obtained from frozen cells [8] and precipitated with 33.33% ammonium sulfate.
The protein pellet was resuspended in 10 rnM Tris buffer, pH 7.3, and incubated with
[3H]muristerone A (30 nM). An aliquot of the radiolabeled receptor preparation was then
subjected to DEAE anion exchange HPLC chromatography. (Chromatographic conditions:
Pharmacia Mono Q HR 515 anion exchange column; 1 ml/rnin; time 0 elution buffer was 50
rnM Tris, pH 8.0, followed at 2 rnin by an 11 rnin 0 to 400 m M KCI gradient in 50 rnM Tris and
at 17 rnin by final elution with 1.0 M KCI in 50 rnM Tris.) Absorbance was monitored at 280
nm (-).
Fractions of 0.5 m l were collected and counted for radioactivity (x-x).
when bound to the ecdysteroid receptor, was much less sensitive to dissociation with high salt (data not shown). In addition, saturation studies, using
[3H]muristerone A as the radioligand, indicate that muristerone A has an
affinity for the ecdysteroid receptor (Kd 4 nM) that is similar to ponasterone
A. Therefore, current efforts in our laboratory are being directed toward
using the [3H]muristerone A as a radioligand to monitor receptor binding
activity while attempting protein separation techniques that utilize high salt
conditions. The data shown in Figure 7 document one such attempt of an
HPLC anion exchan e separation of the 4 S form of the K, cell cytosol
receptor. Using the [ Hlmuristerone A, it now seems probable that a 10,000fold purification of the ecdysteroid receptor can be achieved and subsequent
monoclonal antibodies to the receptor generated.
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Purification of Ecdysteroid Receptors
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measurements, purification, receptors, drosophila, ecdysteroids, alternative, ligand, cells
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